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Papers In Press, published online ahead of print October 26, 2001
J. Biol. Chem, 10.1074/jbc.M108861200
Submitted on September 13, 2001
Revised on October 26, 2001
Accepted on October 26, 2001

Vesicular and non-vesicular sterol transport in living cells: the endocytic recycling compartment is a major sterol storage organelle

Mingming Hao, Sharron X. Lin, Ola J. Karylowski, Daniel Wüstner, Timothy E. McGraw, and Frederick R. Maxfield

Biochemistry, Weill Medical College of Cornell University, New York, NY 10021

Corresponding Author: frmaxfie{at}med.cornell.edu

We examined the intracellular transport of sterol in living cells using a naturally fluorescent cholesterol analog, dehydroergosterol (DHE), which has been shown to mimic many of the properties of cholesterol. Using DHE loaded on methyl-b-cyclodextrin, we followed this cholesterol analog in pulse-chase studies. At steady state, DHE co-localizes extensively with transferrin (Tf), a marker for the endocytic recycling compartment (ERC), and redistributes with Tf in cells with altered ERC morphology. Expression of a dominant-negative mutation of an ERC-associated protein, mRme-1 (G429R), results in the slowing of both DHE and Tf receptor return to the cell surface. [3H]cholesterol is found in the same fraction as 125I-Tf on sucrose density gradients, and this fraction can be specifically shifted to a higher density based on the presence of horseradish peroxidase-conjugated Tf in the same organelle. While vesicular transport of Tf and efflux of DHE from the ERC are entirely blocked in energy depleted cells, delivery of DHE to the ERC from the plasma membrane is only slightly affected. Biochemical studies performed using [3H]cholesterol show that the energy dependence of cholesterol transport to and from the ERC is similar to DHE transport. We propose that a large portion of intracellular cholesterol is localized in the ERC, and this pool might be important in maintaining cellular cholesterol homeostasis.


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